Study On Preparation Method And Anti-allergic Activities Of Rare Ginsenosides

Ginsenosides are the main compounds in the plants of Araliaceae,Panax genus,including Panax ginseng C.A.Mey.,Panax notoginseng?Burk.?F.H.Chen and Panax quinquefolium L.,etc.It has been reported that ginsenosides have various pharmacological activites,such as immunoenhancement,central nervous system regulation,protection of cardiovascular and cerebrovascular system,antitumer and antifatigue activities.Ginsenosides are classfied into major ginsenosides and rare ginsenosides.Major ginsenosides are ginsenosides with high contents in herbs of Panax genus,such as ginsenoside Ra₁,Ra₂,Ra₃,Rb₁,Rb₂,Rb₃,Rc,Rd,Re and Rg₁.Rare ginsenosides are secondary ginsenosides with low or none contents in herbs,such as ginsenoside 20?R/S?-Rg₃,20?R/S?-Rh₂,20?R/S?-Rh₁,Rk₁,Rg₅,Rk₂,Rh₃,F₄,Rg₆,Rk₃ and Rh₄,which could be obtained by the manner of deglycosylation or structure changes in side chains from major ginsenosides.As reported,compared with major ginsenosides,rare ginsenosides showed better performances in antitumer,antifatigue and oxidation resistance.However,there are only a few reports about anti-allergic activity of rare ginsenosides,much more,there are no reports on development of ginseng extract and ginsenosides as new Chinese herbal drug.At present,acid catalytic degradation,alkali catalytic degradation,enzyme degradation and microbial degradation are applied in the preparation of rare ginsenosides.However,there are many disadvantages in these methods,such as lower efficiency,more by-products,higher cost and more pollution in environment.Therefore,study on new preparation method and anti-allergic activities of rare ginsenosides is essential to both academic research and the development of new medical uses of rare ginsenosides.In this work,a new preparation method was developed to obtain ginseng extract and rare ginsenoside monomers.Much more,in order to clarify the activity of rare ginsenosides on allergic diseases,OVA-induced allergic asthma model,antigen-induced RBL-2H3 cells degranulation model and antigen-induced Th1/Th2immune imbalance model were applied.The research contents are as follow:First of all,acid catalytic degradation was applied to prepare ginseng degradation products.Silica gel column chromatography,preparative high performance liquid chromatography and NMR were used to isolate and identify 15 kinds of rare ginsenosides.The rare ginsenosides used as standards,we established HPLC detection method and investigated methodology.The results showed good linear relationship?R²>0.9990?,intra-day precision?RSD<3%?,inter-day precision?RSD<4%?,stability?RSD<3%?and repeatability?RSD<3%?of 15 rare ginsenosides.The recoveries were in the range from 94.11%to 113.15%?RSD<3%?.The ratios of material to solvent,extraction temperature and extraction time were investigated in order to increase the yields of rare ginsenosides under high temperature and high pressure extraction based on the above quantitative method.According to the optimization results,high temperature and high pressure method was established to obtain ginseng extract.1:12 as the ratio of material to solvent,145?as the extraction temperature,2 hours as the extraction time were confirmed as the final optimal condition.Under the conditions,we obtained the ginseng extract which contained 15 kinds of rare ginsenosides,such as ginsenoside Rk₃,Rh_4,20?S?-Rg₂,20?S?-Rh₁,20?R?-Rh₁,Rg₆,F₄,Rk₁,Rg₅,20?S?-Rg₃,20?R?-Rg₃,20?S?-Rh₂,20?R?-Rh₂,Rk₂ and Rh₃.Among them,ginsenoside 20?S?-Rg₂,20?S?-Rh₁,20?R?-Rh₁,Rk₁,Rg₅,20?S?-Rg₃,20?R?-Rg₃ showed higher contents.Compared with the extract prepared by reflux method,the yield of rare ginsenosides in the ginseng extract was 3.93 times higher and the total content of rare ginsenosides was 14.74 times higher.After further purification,the total content of rare ginsenosides in the ginseng extract?GE?was more than 50%.Secondly,OVA-induced allergic asthma model was applied to evaluate the anti-allergic effects of GE.Lung histopathology,immune organ weights,the ratios of CD4⁺to CD8⁺T cells in spleen,total IgE levels in serum and cytokines in bronchoalveolar lavage fluidalveolar lavage fluid?BALF?were investigated respectively.The results showed that GE may have effects in alleviating the inflammatory symptoms in lung tissue,suppressing the increasing tendency of spleen weights and decreasing tendency of thymus weights,suppressing the decreasing tendency of the ratios of CD4⁺to CD8⁺T cells in spleen,the total IgE levels in serum and the IL-4 levels in BALF.Thirdly,in order to clarify the active rare ginsenoside monomers in GE,anti-allergic effects of 8 protopanaxadiol-type rare ginsenoside monomers?ginsenoside Rk₁,Rg₅,20?S?-Rg₃,20?R?-Rg₃,20?S?-Rh₂,20?R?-Rh₂,Rk₂ and Rh₃?were investigated through antigen-induced RBL-2H3 cells degranulation model.After optimized,the concentration of anti-DNP IgE antibody?0.125?g/mL?,the concentration of the DNP-BSA?0.125?g/mL?,and stimulation time?1 h?were confirmed.The results show that ginsenoside Rg₅ and Rh₃ have significant inhibitory effects on the release of?-HEX during the degranulation of RBL-2H3 cells.Ginsenoside 20?S?-Rh₂ have weak inhibitory effects on the release of?-HEX,while the residual rare ginsenosides have no obvious activity.Further investigations also show that ginsenosides Rg₅ and Rh₃ may inhibit the release of?-HEX by inhibiting the extracellular Ca²⁺influx in the mast cells.Ginsenoside Rg₅ is the dehydration product of ginsenoside 20?S?-Rg₃ at C20-22 position,while ginsenoside Rh₃ is the dehydration product of ginsenoside 20?S?-Rh₂ at C20-22 position.Their anti-allergic activities are both stronger than those of the material ginsenosides,suggesting that the double bond at C20-22 position may play a key role in inhibiting the release of?-HEX from mast cells.Fourthly,in the above experiments,ginsenoside 20?S?-Rh₂ had a weak inhibitory effect in the release of?-HEX from mast cells,while ginsenoside 20?R/S?-Rg₃ had no effects.However,it has been reported that ginsenoside Rh₂ and Rg₃ have potential anti-allergic activities.Considering that ginsenoside 20?S?-Rh₂ and 20?R/S?-Rg₃ may play an anti-allergic role through other pathways,antigen-induced Th1/Th2 immune imbalance model was applied,and effects of Langerhans cell-like dendritic cells?LDCs?treated with ginsenoside 20?S?-Rh₂ and 20?R/S?-Rg₃ on Th1/Th2 immune imbalance were investigated.The effects of rare ginsenosides-treated LDCs on expression of Th1/Th2 cytokines and chemokine receptors in mice popliteal lymph node cells were investigated.Then the specific effects of rare ginsenosides on the functions of LDCs were investigated further.The results show that ginsenoside20?S?-Rh₂ and 20?R/S?-Rg₃ both have effects on Th1/Th2 immune imbalance.Ginsenoside 20?S?-Rh₂ could induce differentiation of T cells into Th1 direction by up-regulating the expression of CD40 on LDCs surface,thus regulating the Th1/Th2immune.Ginsenoside 20?R/S?-Rg₃ mainly inhibit differentiation of T cells into Th2direction by down-regulating the expression of TIM-4 on LDCs surface,thus regulating the Th1/Th2 immune,and ginsenoside 20?R?-Rg₃ shows a stronger activity.In summary,a new method for preparing GE by high temperature and high pressure was established in our work firstly.The rare ginsenosides in the extract was qualitatively and quantitatively analyzed.Anti-allergic effects of GE were confirmed through OVA-induced allergic asthma mouse model.Moreover,a new effective part of Chinese traditional medicine extracted from ginseng which has anti-allergic activity has been obtained.Then,the antigen-induced RBL-2H3 cells degranulation model and antigen-induced Th1/Th2 imbalance model were applied to further clarify the rare ginsenoside monomers which play an anti-allergic role,and relative mechanisms were pramiraly studied.The results of this study provide a new preparation method for ginseng extract and rare ginsenosides which have anti-allergic activity,and also provide a new scientific basis for the application of rare ginsenoside in anti-allergic diseases.